De Novo Transcriptome Analysis in Hevea brasiliensis to Unveil Genes Involved in Low Temperature Stress Response

Low temperature is one of the adversities threatening the growth and development and reduces the yield of rubber trees.However,molecular mechanisms toward rubber trees in response to low temperature are largely unclear.In this study,7,159 and 7,600 differentially expressed genes(DEGs)were identified in‘Reyan 73397’rubber trees.Through GO analysis,the catalytic activity was the representative of the GO term in the only DEGs at the two studied temperatures(room temperature and 4°C,respectively),while KEGG analysis showed that carbon metabolism was the most important grouping under the comparison of these two temperatures.In addition,expression of 9 members of transcription factor MYB family genes were further verified by qRT-PCR,and MYB family genes may play important roles in the regulation of rubber trees under low temperature stress.This study provided a theoretical foundation for(1)revealing the molecular mechanisms of rubber trees in response to low temperature and(2)breeding of tolerant varieties of rubber trees.

Localized Effects of Mechanical Wounding and Exogenous Jasmonic Acid on the Induction of Secondary Laticifer Differentiation in Relation to the Distribution of Jasmonic Acid in Hevea brasiliensis

The relationship between the effect of exogenous jasmonic acid (JA) on the induction of secondary laticifer differentiation and the distribution of JA in the seedling of Hevea brasiliensis Mull. Arg. was investigated with the aid of experimental morphological and radioisotope technique. Most radioactivity of H-3-JA sustained in treated site within one hour while no radioactivity was detected in new shoot and the radioactivity in upper leaf was much less than that in the parts below the treated site, suggesting that JA was mainly transported downwards in the shoot of H brasiliensis. Mechanical wounding hindered the entrance of exogenous JA remarkably while held back the entered JA to the regions around wounded site. The effect of exogenous JA and mechanical wounding on the induction of the secondary laticifer differentiation was limited to treated site where high level of JA was expected. Mechanical wounding reduced the effect of exogenous JA on the differentiation of secondary laticifer, which could be ascribed to the hindrance of mechanical wounding to the entrance of exogenous JA. It was concluded from the combined data that a high accumulation of JA was required for inducing the secondary laticifer differentiation in H. brasiliensis.

不同树龄橡胶(Hevea brasiliensis)林细根生物量的垂直分布和年内动态

用根钻法对幼树期（5a）、初产期（9a）和旺产期（16a）3个树龄橡胶树细根（〈2 mm）生物量变化、垂直分布及动态变化进行研究。结果表明：5、9、16a橡胶林0～60cm土层内细根总生物量分别为2056.18、1557.42、1174.90 kg·hm^-2。细根的生物量与橡胶树不同生长或生产期有密切的关系,但幼龄橡胶树细根生物量还容易受环境的影响而改变。橡胶树细根总生物量80%左右分布在0～40cm土层,细根生物量随着土层深度的增加而减少,回归模型分析以指数方程y=a.ebx（a、b均为常数）拟合效果较好。通过不同月份细根生物量比较,发现橡胶树细根生物量年动态变化呈“双峰型”,5a、9a橡胶树峰值分别出现在4月份和8月份;16a橡胶树则出现在6月份和10月份,对环境因子的响应具有一定的滞后性。不同树龄橡胶树表层细根生物量年内变化不一致,其它土层细根生物量年内变化也呈“双峰型”,土层越深,变化越趋于缓慢。

AFLP Fingerprinting Analysis of Elite Hevea brasiliensis Germplasm

There are more than 6 000 clones of Hema brasiliensis Mull. Ary germplasm in the germplasm garden of Chinese National Key Biotechnology Laboratory for Tropical Crops and some of them are elite germplasm demonstrated by production and previous studies. AFLP (amplified fragment length polymorphism) fingerprinting analysis was performed on 25 clones (15 of Wickham clones and 10 of Amazon wild clones which possess phenotypes with high-yielding,/Iow-yielding, cold-tolerance/cold-sensitivity, oidium-resistance/oidium-sensitivity, tapping panel dryness (TPD) /healthy) respectively through a 377 DNA sequencer (P. E. Corp.) and PAGE electrophoresis results were analyzed by using GeneScan(TM) and Genotype(TM) Analysis software (P. E. Corp. The fragment profiles of different clones were obtained. Five hundred and eighteen fragments were generated by two primer combinations screened from 64 primer combinations and 511 fragments appeared to be polymorphic (98.6%). Genetic distance ranged from 0.25 to 0.81 between clones and ranged from 0.07 to 0.17 within RRIM600 clone. A specific 320 bp fragment of the oidium-resistant clones was found through genotype analysis. These results showed that AFLP fingerprints were highly reproducible and powerful and can be widely used in germplasm identification and genetic diversity analysis of Hema brasiliensis. In addition, based on the AFLP data, cluster analysis was performed. Cluster results showed that all the clones studied were almost clustered into a group one by one.

巴西橡胶树(Hevea brasiliensis)微管相关蛋白全长cDNA克隆及表达分析

从巴西橡胶树Heveabrasiliensis差减cDNA文库中分离到微管相关蛋白(Microtubule-associatedprotein,MAPs)基因片段,根据该基因片段序列信息,设计特异引物,采用cDNA末端快速扩增技术RACE(RapidAmplificationofcDNAEnds)进行差异片段的5’和3’端的扩增,获得了长度为788bp的全长cDNA,该基因在GenBank中的登录号为AY461412。序列分析表明该基因包含完整的开放阅读框,编码144个氨基酸,与微管相关蛋白基因家族具有很高的同源性,推测该基因是微管相关蛋白基因。半定量RT-PCR检测证实它在胶乳中的表达强于叶中,胁迫处理(伤害及乙烯处理)使其表达上调。

Fluctuation of Microfibrillar Protein Level in Lutoids of Primary Laticifers in Relation to the 67 kD Storage Protein in Hevea brasiliensis

Abundant microfibrillar protein inclusions were present in the lutoids of the primary laticifers in Hevea brasiliensis Mull. Arg. Two forms of the inclusions could be distinguished under the electron microscope, each in separate lutoids. As revealed by SDS-PAGE, the 59.5 kD and 63.5 kD proteins were the major components of the microfibrillar protein purified by isoeleettic point precipitation. Western-blotting analysis indicated that they were immunorelated with the 67 kD protein accumulated in the protein-storing cells. The 59.5 kD and 63.5 kD proteins were abundant in the uppermost part, the stem of new shoot and sustained their abundance during the growth and development of new shoot while their contents decreased remarkably in the lower parts of the trunk, accompanying by the accumulation of 3-5 kinds of proteins with low molecular weights. This fluctuating pattern suggested that the degradation of the 59.5 kD and 63.5 kD proteins had nothing to do with the new shoot growth and may be closely related to the primary laticifer differentiation. The 67 kD protein could not be detected in the young stem of new shoot when its leaves were broze-colored while the protein started to be accumulated in the stem, when the leaves of new shoot had matured, behaving like a typical vegetative storage protein.

橡胶树(Hevea brasiliensis Muell.Arg.)幼茎木材的超微结构观察

橡胶树是中国重要的热带经济作物,橡胶种植的副产物橡胶木是我国木材供应的重要来源。我们以不同发育阶段的橡胶树幼茎木材为材料,借助扫描电子显微镜技术,对木质部细胞的超微结构进行了观察。结果表明,在橡胶树幼茎木材中,导管和木纤维细胞壁随着木质部发育成熟会发生明显的次生加厚,加厚方式主要为螺纹加厚;木质部各类型细胞均存在大量纹孔,纹孔排列方式主要有散生、网状、梯状和单串状等类型;在木质部发育过程中,木射线和部分薄壁细胞中会逐渐积累大量淀粉粒;木质部细胞内壁及其填充物表面存在不同类型的附着物。研究结果将为橡胶木材材性及其形成机制的研究提供一定理论参考。

Effects of Micro-tapping with Different Concentration of Ethylene on Yield and Physiological Features of Rubber Tree(Hevea brasiliensis) Clone PR107

In this study, the rubber tree clone PR107 was selected as test material,and its yields and physiological features under micro-tapping with different concentrations of ethylene were compared. The results showed that there was no significant correlation between rubber yield and ethylene concentration applied by microtapping on PR107. Too high level of ethylene was not conducive to improvement of rubber yield. When the ethylene concentration for micro-tapping was higher than60%, physical deficiencies appeared in rubber tree PR107, the dry rubber content was reduced, and the yield was unstable. Instead, when the ethylene concentration was lower than 40%, the physiological features of PR107 were steady, the dry rubber content was stable, and the yield was relatively high.

Study on the Explant Disinfection of Root Culture of Hevea brasiliensis

[Objective] The aim was to look for an effective way of decreasing explants contamination in the root tissue culture of Hevea brasiliensis.[Method] In order to study root tissue culture of H.brasiliensis,new roots from forest section after treatment for one month were as explants,and 1 g/L carbendazim,75% ethanol,0.1% mercuric chloride and Yipeilong with different concentrations were used as disinfectants for roots disinfection.[Result] Before conventional disinfection,root explants were treated by 1 g/L carbendazim for 2.5 h,and disinfected by 75% ethanol for 30 s and 0.1% HgCl2 for 6 min,then cultured on the callus induction medium supplemented with 0.1% Yipeilong.The results showed that the contamination rate of explants decreased to 44.59%,and 25.60% explants survived after cultured for 30 d.[Conclusion] The study could provide theoretical foundation for the decrease of explant contamination in the root tissue culture of H.brasiliensis.

Molecular Cloning and Bioinformatical Analysis of a cDNA Encoding Mitochondrial 50S Ribosomal Protein L21 from Hevea brasiliensis Muell. Arg.

[Objective] ＂Tapping panel dryness （TPD）＂, a syndrome known as tapping incision blocked partly or entirely during latex exploiting, has become the most important factor causing great losses for rubber production. Aiming to elucidate the molecular mechanism of tapping panel dryness occurrence, this study carried out molecular cloning and bioinformatical analysis of a mRPL21 cDNA sequence, a gene associated with TPD. [Method] In a preliminary study, an expressed sequence tag （EST） encoding a deduced protein homologous to mitochondrial 50S ribosomal protein L21 （mRPL21）, which showed to be down-regulated in the latex of TPD-affected rubber trees, was isolated by suppression subtractive hybridization （SSH）. After ESTs assembling and RT-PCR validation, an 853 bp cDNA sequence with an open reading frame （ORF） was cloned, which was named as HbmRPL21 under GenBank accession number of HM230670. [Result] Bioinformatical analysis suggests that HbmRPL21 encodes a deduced polypeptide of 271 amino acids with a theoretical molecular weight （Mw） of 30.52 kDa and isolectric point （pI） of 8.40, and HbmRPL21 is a mitochondrion-targeted protein with a conserved domain of Ribosomal_L21p involving translation. Homology analysis reveals high amino acid sequence identity of mRPL21 from plants, while diversity of that between plant and animal kingdom. [Conclusion] This study laid the basis for further revealing the biological functions of mRPL21 in TPD-affected rubber trees.

Evaluation of unconstrained and constrained mathematical functions to model girth growth of rubber trees (Hevea brasiliensis) using young agemeasurements

No attempt has been made to date to model growth in girth of rubber tree （Hevea brasiliansis）. We evaluated the few widely used growth functions to identify the most parsimonious and biologically reasonable model for describing the girth growth of young rubber trees based on an incomplete set of young age measurements. Monthly data for girth of immature trees （age 2 to 12 yearsi from two locations were sub- jected to modelling. Re-parameterized, unconstrained and constrained growth functions,of Richards （RM）, Gompertz （GM） and the monomo- lecular ＇model ^（MM） were fitted to data. Duration of growth was the firsf constraint introduced. In the stagel We attempted a population aver- age （PA） model to capture the trend in growth. The best PA model was fitted as a subject specific （SS） model. We used appropriate error vari- ance-covariance structure to account for correlation due to repeated measurements over time. Unconstrainecl functions underestimated the asymptotic maximum that did not reflective carrying capacity of the locations. Underestimafions were attributed to the partial set＇ of meas- urements made during the early growth phase of the trees. MM proved superior to RM and GM. In the randomcoefficient models, both Gf and Go appeared to be influenced by tree level effects. Inclusion of diagonal definite positive matrix removed the correlation between random effects. The results were similar at both locations. In the overall assessment MM appeared as the candidate model for studying the girth-age relationships in Hevea trees. Based on the fitted model we conclude that, in Hevea trees, growth rate is maintained at maximum value at to, then decreases until the final state at dG/dt 〉 0, resulting in yield curve with no period of accelerating growth. One physiological explanation is that photosynthetic activity in Hevea trees decreases as girth increases and constructive metabolism is larger than destructive metabolism.

Preliminary Study on Seedling Growth Rhythm and Grey Correlation Analysis of Rubber (Hevea brasiliensis) Seedlings in Danzhou District, Hainan

The rubber tree physiological and ecological process quantitatively described by using mathematical method is an important means to the analysis of rubber tree growth process and mechanism. The study on growth simulation model of rubber tree will lay the foundation for the application of rubber tree cultivation intelligent decision system. A Richards equation was formulated to describe the height and stem diameter growth dynamics of the annual rubber seedlings. An area correlation analysis was done according to the closeness of the observed parameters to the dynamic curve on the gray system composed of the seedling growth increment and the meteorological factors including aerial temperature, precipitation and solar radiation hours that influence upon the seedling growth. The results showed that rubber seedling response fitted the Richards equation quite well. The growth increment displayed a distinct alternation of 'slow—fast—slow— fast—slow' rhythm. The growth course of the seedlings might be partitioned into three periods of time by the sequential clustering analysis, namely pre-growing, fast-growing, late-growing stage. The tray correlation analysis revealed that air temperature had the most significant influence while precipitation had the least impact on height growth of the rubber seedlings. In conclusion, the air temperature had the most significant influence while solar radiation hours had the least impact on stem diameter growth of the rubber seedlings.

Effect of natural and synthetic antioxidants on oxidative stability of FAMEs obtained from hevea brasiliensis

Biodiesel(fatty acid alkyl esters),an alternate to fossil fuel,has the tendency of autoxidation and hence requires antioxidants for long term storage.The influence of synthetic and natural antioxidants on the oxidative stability was analysed for fresh FAMEs(fatty acid methyl esters)obtained from hevea brasiliensis at 140 C.Higher activity was observed for synthetic antioxidants following the order of GA】BHT】DTBP】Q】GT-M】PH-M】GT-C,whereas the oxidative stability of stored FAMEs samples measured at 110 C reveals a nearly inverse trend.Storage stability was tested for the FAMEs obtained from hevea brasiliensis stored at 30 C,after addition of synthetic and natural antioxidants—butylated hydroxytoluene(BHT),2,6-di-tert-butylphenol(DTBP),quercetin(Q),gallic acid(GA),methanol extracts from green tea(GTM),pomegranate hull(PH-M),and chloroform extract of green tea(GT-C).Antioxidant activities above 1500 ppm was in the order of DTBP】BHT】GA】GT-C】GT-M】Q】PH-M.Synthetic antioxidants have been found more efficient to improve the storage stability of FAMEs obtained from hevea brasiliensis.DTBP in particular has the highest protection factor.

Molecular Cloning and Analysis of LEA14L2 Gene from Rubber Tree( Hevea brasiliensis Muell. Arg. )

Using an isotig encoding a putative polypeptide with high similarity to Arabidopsis LEA14 as a query, a 613 bp long cDNA was in silico cloned from the transeriptome data of rubber tree. The sequence nominated as HbLEA14L2 contains an ORF of 456 bp with 3 bp 5＇ UTR and 154 bp 3＇ UTR. Subsequently, a 464 bp eDNA and an 834 bp genome sequence containing this ORF was amplified and sequenced. Sequence analysis suggested that HbLEA14L2 has one intron and encodes 151 amino acids with a theoretical molecular weight of 16.55 kDa, isoleetric point of 4.93 and GRAVY value of -0.022, indicating a cytoplasmle localization pattern; HbLEA14L2 protein contains a conserved LEA_2 domain and belongs to LEA_2 subfamily, sharing 91%, 76%, 75%, 72% and 63% similarity with the homologous proteins in castor bean, leafy spurge, poplar, cotton, and Arabidopsis, respectively. Swiss-Model indicated that the tertiary structure of HbLEA14L2 is consisted of one α-helix and seven β-sheets, which was proposed to serve as a regulatory protein to prevent cellular desiccation.

Intra-and inter-clonal tree growth variations of Hevea brasiliensis

We evaluated the effects of planting densities （500, 1,000, 1,500 and 2,000 trees.ha-1） on tree growth performance （diameter at base, diameter at breast height, and clear bole height） of two clones （RRIM 2020 and PRIM 2025） of nine years old plantations of rubber tree （Hevea brasiliensis Muell. Arg） in Malaysia. For the four planting densities of the two clones, basal area and diameter at breast height declined with increasing planting density. Clear bole heights were greatest at 1,500 trees.haland lowest at 500 trees.ha-1 for the clone RRIM 2020, and at 2,000 trees.ha-1and 500 trees.halfor clone RRIM 2025. We conclude that the ideal planting density is 2,000 trees.ha＂l for obtaining high volume of wood production and 500 trees-ha-1 for high wood quality.

An Analysis of the Patents concerning Hevea brasiliensis

This paper mainly analyzes the application data of patents concerning Hevea brasiliensis in the world and China from the perspectives of time,research field,and the geographical distribution of applicants. It also interprets the patents for invention in China from time,research field,and types of applicants. Based on the study,it shows that although the patent applications fluctuate,the general trend is rising. And the patents are mostly concentrated in plant protection,and agricultural biotechnology. The Chinese applicants of patents for invention largely work in the research institutes,and most of these patents focus on the field of plant protection,agricultural biotechnology,and agro-processing.However,the foreign applicants mainly work in companies,and most of these patents only focus on plant protection. These findings will provide a reference for formulating development policies about natural rubber industry in China.

Molecular Cloning and Sequence Analysis of an Aquaporin Gene Related to Tapping Panel Dryness in Rubber Tree( Hevea brasiliensis Muell. Arg. )

As a syndrome with tapping incision blocked partly or entirely during latex exploiting in rubber tree （Hevea brasiliensis ）, ＂Tapping panel dryness （TPD）＂ causes great yield losses, thereby becoming the most important factor limiting rubber production. On the basis of an EST down-regulated in TPD-affected rubber trees, a 903 bp cDNA denoted HbPIP2;2 was isolated from the bark tissue with a combination of in silico cloning and RT-PCR. The cDNA contains an 867 hp ORF, 13 bp 5＇UTR and 23 bp 3＇ UTR. Sequence analysis indicated that HbPIP2;2 encodes 288 amino acids with a theoretical molecular weight （Mw） of 30. 71 kDa and isolectric point （Pi） of 8.20. Bioinformatics analysis suggested that the deduced protein is predicted to have six transmembrane helices located to the plasma membrane and harbor one conserved MIP domain that can be grouped into plasma membrane intrinsic proteins （PIPs） of aquaporin （AQP） family. Homolo- gy search revealed that the protein shares a similarity of more than 90% with the homologues in Ricinus communis, Popttlus trichocarpa, Juglans regia and Theobro- ma cacao, suooorting a hie.hly conserved evolution. This study provided basis for further uncovering the regulatory role of AOPs in TPD occurrence.

Advances in Tissue Culture of Rubber( Hevea brasiliensis L. )

Natural rubber, which is produced as a secondary plant metabolite, has been well recognized as an important raw industrial material due to its unique properties that can not be substituted by any alternative synthetic materials. Although more than 2000 plant species have been shown to possess the ability to synthe- size natural rubber, Hevea brasiliensis L. is yet the only one qualified as a commercial source for natural rubber production, which not only has important economic value, but ＂also leads to significant ecological and social benefits to maintain ecological forest microclimate and ease current ecological issues caused by dwindling natural forests. With the growing demand for natural rubber, providing a large number of high-quality rubber planting materials has become a major issue to be solved. In this paper, advances on tissue culture of H. brasiliensis were summarized, which laid the foundation for breeding high-quality rubber planting materials.

Molecular Cloning and Sequence Analysis of a Tonoplast Aquaporin Gene TIP1 Associated with Tapping Panel Dryness in Hevea brasiliensis

[Objective]To address the role of aquaporins（AQPs）in the occurrence of Tapping Panel Dryness（TPD）,a phenomenon with tapping incision blocked partly or entirely during latex exploiting in the rubber tree（Hevea brasiliensis）that causes great losses on rubber production,a tonoplast aquaporin gene associated with TPD occurence was cloned and analyzed.[Method]Based on an EST down-regulated in TPD-affected rubber trees,a 774 bp cDNA designated HbTIP1 was isolated from the bark tissue of Hevea brasiliensis with a combination of in silico cloning and RT-PCR.And the gene structure and sequence characteristics were analyzed using bioinformatics tools.[Result]The cDNA includes a 759 bp ORF,8 bp 5’UTR and 7 bp 3’UTR.Sequence analysis indicated that HbTIP1putatively encodes 252 amino acids with a theoretical molecular weight（Mw）of 25.88 kDa and isolectric point（pI）of 4.96.Bioinformatics analysis suggested that the deduced protein is predicted to have six transmembrane helices located to the vacuolar membrane and to harbor one conserved MIP domain that can be grouped into the tonoplast intrinsic proteins（TIPs）of aquaporin family.Homology search revealed that the protein shares a similarity of more than 90%with the homologues in Theobroma cacao,Prunus persica,Citrus sinensis and Ricinus communis,supporting a highly conserved evolution.[Conclusion]This study provided basis for further revealing the regulatory role of AQPs in the TPD occurrence.

Studies on Rubber (<i>Hevea brasiliensis</i>) Trees Exist Plant Type after Planting and Available Tapping Tree of Rubber Plantation in China

Existing plant types of rubber tree after planting and available tapping tree were investigated, and there were about 28 rubber plantations with different tapping years of 8 varieties “CATAS7-33-97”, “CATAS8-79”, “CATAS7-20-59”, “PR107”, “RRIM600”, “GT1”, “INA873”, “93-114”in South China. The results showed that there were six kinds of existing plant types of rubber tree after planting of rubber plantations, which were available tapping trees, wind damaged trees, cold damaged trees, tapping panel dryness trees, absent trees and weak trees, respectively. These data investigated also showed rubber trees under available tapping, stoppage due to tapping panel dryness, absence, wind damage, cold damage and weakness were counted and calculated and made up for 72.21%, 14.75%, 5.61%, 3.86%, 2.68% and 1.89%. Tapping panel dryness trees, wind damage and absent trees are major factors for the loss of tapping rubber trees in the rubber plantations. Of these investigated varieties, available tapping trees per 100 trees of rubber plantation of “PR107”at the 1st, 12th, 14th, 16th, 20th, 24th tapping year were 96, 67, 70, 75, 66, 46 trees in Hainan planting zone, respectively. Available tapping trees per 100 trees of rubber plantation of “RRIM600”at the 9th, 15th, 20th, 22nd tapping year were 88, 62, 55, 36 trees in Yunnan planting zone, respectively. Available tapping trees per 100 trees of rubber plantation of “93-114” at the 10th, 19th, tapping year were 94, 62 trees in Guangdong planting zone. These results showed that available tapping trees of rubber plantation decreased with increasing tapping age under different planting zones in China.